PROJECT SUMMARY/ABSTRACT RNA modifications influence RNA expression and function without changing the underlying nucleotide sequence. N6-methyladenosine (m6A) is the most abundant mRNA internal modification, existing in thousands of mRNAs, of which the majority encode for regulatory proteins such as transcription factors. Although emerging evidence links m6A to stem cell differentiation, neurological disease and cancer, the molecular and regulatory mechanisms of m6A are poorly understood. To begin to address this significant knowledge gap, I previously developed the m6A-CLIP method to precisely map m6A modifications in mRNAs genome-wide, enabling the direct examination of m6A function in a site-specific manner. We discovered that m6A addition is part of mammalian pre-mRNA synthesis. We also showed that site-specific mutation of m6A sites leads to increased mRNA half-life in the cytoplasm. Thus, my work in the last five years has established a completely new role for m6A as an important link between nuclear events that occur on nascent pre-mRNA and cytoplasmic events that determine mRNA stability. These findings provide the premise for the current proposal and they uniquely position my interdisciplinary research program to carry out the proposed studies. Here, our objectives are to identify the molecular mechanisms regulating m6A site-specific deposition and m6A function in cytoplasmic mRNA stability. We will reach these objectives by biochemical characterization of the m6A protein complexes. We will also perform genome-wide CRISPR interference screening to systematically identify m6A regulators. Finally, we will build a data-driven computational model to predict m6A site-specific deposition and its effects in cytoplasmic mRNA turnover. Thus, successful completion of the proposed work will systematically reveal the underlying mechanisms of m6A RNA biology by combining cutting edge computational biology, innovative biochemistry and high-throughput genetics. These findings are expected to have a transformative impact on the RNA field by stimulating further studies into the role of mRNA modifications in regulating gene expression. Our long-term goal is to leverage the mechanistic information learned herein to gain deep insights into the role of m6A in human disease and to design novel mRNA modification-targeted therapeutics that regulate gene expression without altering the host genomic sequence.